Table 1.
List of primers used in quantitative RT-PCR.
Fig 1.
Characteristics of mouse NCSC and MSC from adult bone marrow.
NCSC obtained from Wnt1-Cre/R26R-LacZ mice expressing LacZ. In adherent culture conditions, MSC did not express β-galactosidase (a) neither sox10 (b), but a small and constant proportion of cells slightly expressed nestin (c) and p75NTR (d). NCSC expressed β-galactosidase (e), sox10 (f), Nestin (g) and p75NTR (h). Finally, among these two populations, only NCSC were able to grow as spheres; spheres also express β-galactosidase (i), Sox10 (j), Nestin (k) and p75NTR (l). (Scale bars = 40μm).
Fig 2.
Cell origin and characterization.
BMSC were obtained from iliac crest aspiration performed on three healthy donors (man and women) aged of more or less 20 years old (b). ATSC (a) and NCSC (c) were obtained using residual material form abdominoplasties or breast reductions performed on healthy women from 18 to 52 years old. We compared the ability to grow as sphere and the CFU potential between donors from the same cell type and we did not observed statistically significant differences between them. Statistical analysis: one way ANOVA followed by HSD post hoc test.
Fig 3.
Characterization of human bone marrow, adipose tissue and dermis-derived stem cells, according to the International Society for Cell Therapy criteria.
Mesenchymal and non-hematopoietic proteins expression were studied and quantified using flow cytometry phenotyping. Homogeneous populations of >95% of ATSC, BMSC and SKSC expressed mesenchymal markers: CD73 (a, h, o), CD90 (b, i, p) and CD105 (c, j, q) while the specific expression of hematopoietic markers was restricted to <5% of the cells: CD3 (d, k, r), CD14 (e, l, s), CD19 (f, m, t), CD34 (g, n, u), CD45 (d-g, k-n, r-u), HLA-DR (a, h, o), CD80 (b, i, p) and CD31 (c, j, q).
Fig 4.
Mouse NCSC-related characteristics in human cells.
Similarly to immunostainings performed in mouse (Fig 1), human ATSC, BMSC, SKSC expressed NESTIN (a-c), did not express SOX10 (e-g). ATSC and BMSC express weakly P75NTR (i-j) in comparison to SKSC (k). We also used a cell line derived from human malignant melanoma cells (MeWo), which was a positive control for NC identity (d, h, l). (Scale bars = 20μm).
Fig 5.
Sphere forming ability and properties of human cells.
The majority of ATSC, BMSC and SKSC were able to grow as sphere, in specific culture medium. Using light and transmission microscope, we were not able to distinguish adherent cells from re-adherent sphere-derived cells (a and b). Spheres presented a diameter between 80 and 350 microns (c). We quantified the ability to each cell type to grow as spheres, after 12 days of culture compare to the initial number of plated cells (75 000 cells in 25 cm2 T-flask). SKSC presented a significantly higher ability to grow as spheres compared to BMSC and ATSC (respectively p<0.0001 and p<0.05 one way ANOVA followed by HSD post hoc test) (d). Similar immunostaining characterizations as performed in Fig 4 were performed on sphere-derived cells. In this case, human sphere-derived ATSC, BMSC and SKSC expressed NESTIN (e-h) and did not express SOX10 (m-p). However, unlike adherent cells, sphere-derived ATSC, BMSC and SKSC expressed P75NTR (i-l). Quantitative RT-PCR were performed on adherent and sphere-derived cells and confirmed immunological observation made at mRNA level (h, l, p). Data were normalized to SKSC adherent cells expression level which was set as 1. Statistical analysis: one way ANOVA followed by HSD post hoc test. **** means p<0.0001. (Scale bars = 50μm).
Fig 6.
Differentiation abilities of human bone marrow, adipose tissue and dermis-derived stem cells.
ATSC, BMSC and SKSC were able to differentiate into adipocytes (lipids vacuoles stained with Oil Red-O—a-c), osteoblast (calcium deposits stained with Alizarin Red—d-f), chondrocytes (chondrocyte matrix stained with Alcian Blue—i-k). Osteoblastic and chondrogenic differentiations were also assessed by quantitative RT-PCR based on RUNX2 (g), ALPL (h), AGGRECAN (j) and BARX2 (k) expression levels. Data were normalized to SKSC undifferentiated cells expression level set as 1. Statistical analysis: one way ANOVA followed by HSD post hoc test. * means p<0.05, *** means p<0.0005, **** means p<0.0001. (Scale bars = 50μm).
Fig 7.
Differentiation abilities of human bone marrow, adipose tissue and dermis-derived cells.
ATSC, BMSC and SKSC were able to differentiate into Schwan cells as the majority of the cells were stained with anti-S100β antibody (a-c) and a small amount of S100β+ cells was also stained with anti-MBP (red) and anti-P0 (green) antibodies (d-f). Quantitative RT-PCR for S100β, PMP22, MBP and P0 confirmed the results obtained by immunofluorescences, at mRNA level (g-j). The three cell types were also able to differentiate into melanocytes stained with anti-TYRP1 antibody (k-m), which was confirmed at mRNA level based on quantitative RT-PCR for MITF (n) and TYRP2 (o). Finally, neuronal differentiation was assessed using anti-NeuN (green) and anti-Neurofilament (green) antibodies (p-r). Data were normalized using SKSC undifferentiated cells expression level set as 1. Statistical analysis: one way ANOVA followed by HSD post hoc test. * means p<0.05, ** means p<0.005, *** means p<0.0005. (Scale bars a, b, c, d, e, f, p, q and r = 50μm; Scale bars k, l,and m 20μm).
Fig 8.
Quantitative RT-PCR characterization using stem cells and NCCs-related markers.
The expression of neural crest and stem cells genes was studied here by quantitative RT-PCR. ATSC, BMSC and SKSC either in adherent or in sphere culture condition expressed NCSC markers such as BRN3A, FOXD3, NGN1, PAX3, SLUG, SNAIL1, SOX9 and TWIST and neural differentiation marker such as MSI1. Data were normalized using SKSC adherent cell expression level set as 1. Statistical analysis: one way ANOVA followed by HSD post hoc test. * means p<0.05, ** means p<0.005, *** means p<0.0005, **** means p<0.0001.
Fig 9.
Expression of SOX9 and TWIST by ATSC, BMSC and SKSC.
The SOX9 and TWIST expression which were previously analyzed at mRNA level, were now studied at protein level. It appeared that 25.7% (±0.9%) of adherent ATSC, 20.5% (±2.7%) of adherent BMSC and 29.8% (±2.6%) of adherent SKSC expressed SOX9. Similar results were obtained concerning TWIST: 42% (±2.7%) of adherent ATSC, 33,4% (±3.6%) of adherent BMSC and 44,2% (±1.7%) of adherent SKSC whereas both proteins were expressed by more than 80% of the cells, in spheres. Arrows indicated either SOX9 or TWIST positive cells in adherent culture condition. Statistical analysis: one way ANOVA followed by HSD post hoc test. * means p<0.05. White arrowheads indicate the positive cells. (Scale bars for a, b, c, h, i, and j = 40μm; Scale bars for e, f, g, l, m, n = 50μm).
Fig 10.
Characterization of adherent bone marrow, adipose tissue and dermis-derived stem cell migration abilities when injected into chick embryos: Localization of migrating cells.
Fig 10 represents transversal (a-o) and longitudinal (p) sections of adherent cells injected into HHSt18 chick embryos. Human stem cells derived from adipose tissue, bone marrow and dermis were localized into chick DRG (a-c), boundary cap of the NT (d-f), injection site (g-i), skin or more precisely melanocyte region (j-l) and finally the fiber track leaving the DRG (m-o). Fig 10p presents longitudinal section with magnification on migrating cells along the neural tube. (Scale bars = 50μm, Green: TUJ1 labeling, Red: human nuclei labeling).
Fig 11.
Characterization of migration abilities of bone marrow, adipose and dermis-derived spheres when injected in chick embryos: Localization of migrating cells.
Fig 11 represents transversal sections of spheres injected into HHSt18 chick embryos. Cells originating from human adipose, bone marrow and dermis-derived spheres were localized into chick DRG (a-c), boundary cap of the NT (d-f), injection site (g-i), skin or more precisely melanocyte region (j-l) and finally the fiber track leaving the DRG (m-o). (Scale bars = 50μm, Green: TUJ1 labeling, Red: human nuclei labeling).
Fig 12.
Chemotaxis assay on human adherent ATSC, BMSC and SKSC.
Chemotaxis assay were build to confirmed migration abilities observed in chick embryos. We placed ATSC, BMSC or SKSC on top of a 10 μm-filter, with cocktail of SDF-1, SCF and NT3 in the bottom chamber. After 48 hours, migration rate was evaluated by the quantification of the filter area occupied by human cells. Passive migration was observed and quantified in control condition (only serum-free DMEM in the bottom chamber) and this value was used to normalize the results obtained with different cocktail concentrations (Table). Statistical analysis: (n = 3), one way ANOVA followed by HSD post hoc test. * means p<0.05, ** means p<0.005, *** means p<0.0005, **** means p<0.0001.
Table 2.
Recapitulative table of the ability of human cells from adipose tissue, bone marrow and dermis to grow as spheres and express NCSC markers.
Fig 13.
Summary: NCSC identification in adult human bone marrow, adipose tissue and dermis.
The phenotypic characterization, multipotency and the migration abilities of ATSC, BMSC and SKSC strongly suggest the presence of neural crest-derived stem cells in human adult adipose tissue and bone marrow.